1. Field of the invention
The present invention relates to the isolation of genes encoding polyphenol oxidase (PPO) from plants. More particularly, the present invention provides methods for obtaining genomic DNA encoding PPO enzymes and fragments of a wide range of plants, such as, for example, strawberry, tobacco, apricot, avocado, cherry, peach, pear, coffee, apple, lettuce, French bean, banana, rice, and potato. The present invention clearly extends to the isolated genomic DNAs encoding said PPO enzymes and fragments.
Browning of plant tissues often occurs following injury or damage and this generally results in spoilage of fruit and vegetables. Undesirable browning also occurs during processing of plant materials to produce food or other products. Steps are taken during transport, storage, and processing to prevent these browning reactions. Often this involves the use of chemicals such as sulphur dioxide but the use of these substances is likely to be restricted in the future due to concerns about their safety and consumer acceptance. For example, the US Food and Drug Administration banned the use of sulphite for most fresh fruit and vegetables in 1986. The production of fruit and vegetable varieties with an inherently low susceptibility to brown would remove the need for these chemical treatments.
It will be understood that browning in plants is predominantly catalysed by the enzyme PPO. PPO is localised in the plastids of plant cells whereas the phenolic substrates of the enzyme are stored in the plant cell vacuole. This compartmentation prevents the browning reaction from occurring unless the plant cells are damaged and the enzyme and its substrates are mixed.
It will be apparent from the preceding discussion that there is a need to develop methods for reducing the level of browning of plant tissues. One approach is to modulate the level of expression of PPO genes in plants. A prerequisite to achieving this objective by molecular means is the isolation of nucleic acid encoding PPO genes from a variety of plant sources.
2. Description of Related Art
The prior art includes International Application PCT/AU92/00356 to the present applicant which describes the cloning of PPO genes from grapevine, broad bean leaf, apple fruit and potato tuber. This application recognises that PPO levels in plants may be manipulated by increasing or decreasing expression of PPO gene. The application also identifies two conserved copper binding sites in PPO genes, designated CuA and CuB, and predicts that these regions are suitable for design of probes and primers to obtain other plant PPO genes. However, the method described in PCT/AU92/00356 suffers from the disadvantages that it is necessary to identify the appropriate stage of development of the target tissue, isolate mRNA or total RNA and synthesize cDNA. This in turn requires a relatively large amount of plant material.